Goldenseal An Appalachian Plant Monograph Hydrastis canadensis

Transcription

An Appalachian
Plant Monograph
Chief author and editor: Andrew Pengelly PhD. Assistant authors: Kathleen Bennett MS, Kevin Spelman PhD, Michael Tims PhD. Peer review board: Paul Bergner AHG, Hans Wolmuth PhD. Editorial team: Bevin Clare MS, Mimi Hernandez MS, James Snow MA, Amanda Vickers MS. Citation Instruction: Pengelly, A., Bennett, K., Spelman, K., & Tims, M. (2012). Appalachian plant monographs: Hydrastis canadensis L., goldenseal. Appalachian Center for Ethnobotanical Studies. Cover illustration by Peggy Duke Goldenseal
Hydrastis canadensis L.
1. Taxonomy
Hydrastis canadensis L.
Family: Ranunculaceae (buttercup family)
Hydrastis is a monotypic genus, which some authors have placed in a
separate family – Hydrastidaceae (Tobe & Keating, 1985) – though
more recent genetic studies confirm H. canadensis as the basal branch
of the Ranunculaceae, albeit with close ties to the Berberidaceae family
(Ro, Keener, & McPheron, 1997; Chu, Li, & Qi, 2006).
Common names: golden seal, eyebalm, eyeroot, golden root, ground
raspberry, Indian dye, Indian turmeric, jaundice root, orange root,
yellowroot, yellow pucoon.
APPALACHIAN PLANT MONOGRAPHS
1. Taxonomy
1
2. Botanical description
and distribution
2
3. Traditional use
3
4. Phytochemistry
4
5. Pharmacology and
toxicology
11
6. Clinical studies
15
7. Modern phytotherapy
16
8. Sustainability and
cultivation
17
9. Summary and moving
forward
24
10. References
25
Hydrastis canadensis
2. Botanical description and
distribution
Hydrastis canadensis is an herbaceous perennial growing from a short
yellow rhizome. The rhizome has a knotty appearance bearing
remnants of stems or stem scars (Tobe & Keating, 1985). During the
first year vegetative growth consists of a pair of leaf-like cotyledons on
long petioles. In the second year a few inch long ‘footstalk’ emerges
bearing one palmately-lobed or maple-shaped leaf with biserrate
margins near the apex. Over the next two years a true stem arises
attaining a height of one foot or more, bearing two or three petiolate
leaves arranged alternately on the stem. Small, solitary, greenish-white
flowers (up to 75 stamens enclose a fewer number of carpels with short
styles) appear at the base of the leaves, born on short pedicels. The
flowers develop into a compound fruit consisting of an aggregate of
drupelets, with each drupelet bearing 1-2 seeds. The fruit is inedible to
humans (Krochmal, Walters and Doughty 1969; Upton, 2001).
Detailed macroscopic botanical illustrations from the Botanical Gazette
(Bowers, 1891) are reproduced in Appendix 1.
H. canadensis is an understory plant found growing in patches in rich
open woodlands, hill slopes and along stream banks (Bowers, 1891;
Tobe & Keating, 1985). The natural distribution range includes Ontario
Canada, through New York south to Tennessee and Georgia, and it was
historically abundant in Ohio, Kentucky and West Virginia (McGraw,
Sanders, & Van der Voort, 2003), however it has been experiencing
significant population decline in recent decades (Sanders & McGraw,
2005). Figure 7 illustrates the natural distribution as determined by the
United States Department of Agriculture (USDA).
3. Traditional Use
Traditional use in Appalachia
The root of H. canadensis is highly prized in Appalachian culture as a strengthening tonic, stomachic and as
a source of yellow dye. It is also applied topically for sore eyes and general ulcerations (Millspaugh, 1974;
Crellin & Philpott, 1990). The powder preparation is combined with water and gargled as a sore throat
remedy. Interestingly, it once had a reputation for preventing “pitting” scars from smallpox when applied
topically in a warm bath (Cavender, 2003). A common complaint of Appalachian folk is that of “sour
stomach” or dyspepsia, which in many cases is linked to tooth decay, gum disease with diminished chewing
capabilities. For this H. canadensis, is widely used, either by chewing on or making a decoction of the root
to ease the stomach upset (Cavender, 2003). It has also been used to treat arrow wounds! (Jacobs & Burlage,
1958).
2
Traditional use outside of
Appalachia
Native Americans
H. canadensis was a major medicine for the Cherokee, both as a bitter
tonic and as an eyewash for inflamed eyes (Upton, 2001). It was also
popular with the Iroquois, whose recorded uses include whooping
cough, diarrhea, pneumonia and tuberculosis (Moerman, 1986). More
generally, it was widely used for tumors and also as a dermatologic
aide topically for ulcers and skin disorders (Duke, 1986). H. canadensis
was a common remedy for stagnant gastrointestinal function
characterized by a “sour” stomach (Moerman, 1998).
Folklore & Home
Figure 1. Goldenseal (Hydrastis
canadensis): A. Young plant with
horizontal rhizome and numerous
roots. B. Fruit-head of small
berries. C, older plant showing
the palmately lobed leaves.
Reproduced from Scientific and
Applied Pharmacognosy by
Henry Kraemer (1915).
H. canadensis was used to address dyspepsia and as a tonic for
depleted digestive organs. As a wash it was beneficial for sore,
inflamed eyes, and as a gargle for sore mouth and throat (Gardner &
Aylworth, 1836). The root was generally valued as a household bitter
by adding it to wine or brandy. The root was added to wine or brandy
and valued as a household bitter (Sumner, 2004).
Physiomedicalism
For Samuel Thomson, regarded as the founding father of Physiomedicalism, H. canadensis was a simple bitter, given as a powder in
hot water to treat digestive distress and for “correction of the bile”
(Thomson, 1835). Howard, an MD and follower of Thomson,
recommended H. canadensis for debility (weakness), loss of appetite
and for recovery from fevers (Howard, 1853). Alvin Curtis, known as a
“neo-Thomsonian” and Physiomedicalist, also proclaimed the use as a
digestive bitter and as a treatment for bilious colic, as well an
ingredient in medicated eye washes (Curtis, 1858). William Cook,
(1869) claimed that the root of H. canadensis is most beneficial to the
mucus membranes, digestive tract and uterine organs. Cook praised it
in depleted digestion, “it improves appetite and digestion; and through
the stomach proves one of the most acceptable of all general tonics in
indigestion, feeble assimilation, biliousness, leucorrhea, prolapses, and
all forms of debility” (Cook, 1869, p. 474).
In his classic text Back to Eden first published in 1939, Jethro Kloss
emphasized the special influence of H. canadensis on mucous
membranes and skin, and recommended a tea be used as a wash for
“open sores, inflammations, eczema, ringworm and erysipelas” (1972,
p.244), followed by application of the powdered root and a covering.
For ulceration of the mouth, stomach, duodenum, and for tonsillitis he
3
recommended a tea made with one part goldenseal to
one-fourth myrrh - while for pyorrhea and sore gums
he brushed the teeth and gums with a toothbrush
dipped in the same infusion (Kloss, 1972).
Eclectics
H. canadensis was included in the earliest
publications of the Eclectic school, including
Wooster Beachs’ American Practice of Medicine
(1833). It was regarded as equally beneficial in
catarrhal conditions of the intestines, gall ducts,
biliary passages and in jaundice (Felter & Lloyd,
1898). It was considered to act mainly by increasing
hepatic secretion in the liver, and was also used for
disorders of the mucus membranes. A specific
indication was “catarrhal states of the mucous
membranes unaccompanied by acute inflammation”
Felter (1922). Culbreth (1917) favored its’ use as a
local treatment for gonorrhea, leucorrhea, otorrhea
and numerous other infectious disorders marked by
catarrhal discharges.
The homeopath Edwin Hale provided a major
contribution on the therapeutic uses and indications
for H. canadensis (Hale, 1875). With his frequent
use of tinctures and herbal teas - often in preference
to the potentized homeopathic preparations - Hale
was by definition an Eclectic practitioner with his
blend of homeopathy and medical botany. His
description of the action of H. canadensis on
mucous surfaces influenced all of the practitioner
schools of his day. The mucous secretion for which
the herb is indicated is “at first clear, white,
transparent and tenacious, it becomes yellow, or
thick, green and even bloody, and nearly always
tenacious”. He goes on to explain “its’ secondary
effects are exhaustion or destruction of the glandular
sources of the mucus - a condition in which the
mucus surface is dry, glazed, and its functions
destroyed” (Hale, 1875 p. 313).
Regulars
Allopathic physicians employed H. canadensis as a
stimulating tonic, specific for the digestive tract and
liver function. For example, Potter’s - the famous
4
British compendium of plant medicines– listed it as
an “astringent bitter, promoting appetite and
digestion, increasing the secretions of the gastrointestinal tract, and the flow of bile” (Potter, 1906,
p.62). Reported uses included: rhinitis, gastritis,
vaginitis, and urethritis, and as a bitter for general
debility and digestive impairment. Previously Stille
(1874) questioned the reputations of those who
asserted H. canadensis was effective for such
disorders, claiming they lacked an understanding of
medicine and disease!
Henry Rutherford, assistant-physician at Chelsea
Hospital for Women (U.K.), reported five case
studies involving the internal prescription of H.
canadensis tincture for uterine fibroids in the British
Medical Journal (Rutherford, 1888). Excessive
bleeding associated with the condition was
diminished following the treatments in all cases.
According to Rutherford the hemostatic properties of
the herb were acknowledged in France, Germany,
and especially in America where “the drug has had
extended trials, and the published results are most
satisfactory” (Rutherford, 1888 p. 123).
H. canadensis was included in the United States
Pharmacopoeia between 1830 and 1926 and the US
National Formulary from 1926 to1960 (Upton,
2001).
4. Phytochemistry
Alkaloids
The United States Pharmacopoeial Convention has
defined goldenseal based on alkaloid concentrations,
such that the dried roots and rhizomes comprising no
less than 2% hydrastine and 2.5% berberine (USP,
2003). The British Herbal Compendium
recommends similar values: 1.5 - 4% hydrastine,
2.5% berberine and 0.5% canadine (Bradley, 1992).
Isoquinoline alkaloids were first found in H.
canadensis as early as the 1820s. Initially, a yellow-
Table 1. Isoquinoline alkaloids from H. canadensis
Major alkaloids
Berberine
(—)-(β)-hydrastine
(—)-canadine
Minor alkaloids
Hydrastinine
Canadaline
Isohydrastidine
1-β-hydrastine
5-hydroxytetrahydroberberine
(S)-corypalmine
(S)-isocorypalmine
(S)-tetrahydropalmatine
Berberastine
8-oxotetrahydrothalifendine
Canadinic acid
(Galeffi, Cometa, Tomassini & Nicoletti, 1997;
Govindan & Govindan, 2000; Upton, 2001; Hwang,
Roberts, Chadwick, Wu, & Kinghorn, 2003; Weber
et al., 2003; Brown and Roman, 2008)
colored compound named berberine was isolated
from the European herb Berberis vulgaris L., while
in 1828 Rafinesque discovered a yellow-colored
compound in H. canadensis which he named
hydrastine. This name persisted in the US until the
1860s when Mahla established that the H.
canadensis compound referred to as hydrastine was
the salt of an alkaloid, that alkaloid being berberine
(Lloyd & Lloyd, 1884). The Eclectics, in particular,
were unwilling to forsake the original name
introduced by Rafinesque in favour of the European
designated name (Lloyd & Lloyd, 1884). Lloyd as
pharmacist prepared pure samples of berberine salts
which he found crystallized into dark brown-red
needles, and submitted them to Professors Power
and Coblentz, whose analyses confirmed their
identity and established the formula as C20H17 NO4
(Lloyd & Lloyd, 1884). Berberine is classed as a
quaternary protoberberine alkaloid, occurring
mainly as water soluble cations, and with a very
wide distribution in the plant kingdom (Grycová,
Dostál, & Marek, 2007). Hydrastine is a
phthalidisoquinoline alkaloid, possessing a
tetracyclic nucleus incorporating a γ-lactone ring.
Canadine is a tertiary protoberberine alkaloid,
lacking conjugation in ring C (Wagner, Bladt, &
Zgainski, 1984; Galle, Müller-Jakic, Proebstle,
Jurcic, Bladt, & Wagner 1994; Gocan, Cimpan, &
Muresan, 1996).
The colorless phthalidisoquinoline alkaloids
hydrastine (not to be confused with Rafinesque’s
alkaloid mentioned above) and canadine, were first
isolated from H. canadensis by Perrins in 1862 and
Hale in 1873, respectively (Felter & Lloyd, 1898).
Lloyd’s adaption of Hale’s method for extracting
(the true) hydrastine ‘in a pure state’ from H.
canadensis is described by Professor Power, who
contributed a treatise on the compound to the
American Pharmaceutical Association confirming
the formula as C22H23 NO6 (Power, 1884). Despite
several attempts, Lloyd was unable to confirm the
presence of “Hales’s third alkaloid” as he called the
substance which later became known as canadine
Figure 3. Structures of major alkaloids from H. canadensis. Reproduced with permission
from American Herbal Pharmacopoeia Goldenseal root monograph (Upton, 2001).
5
(Lloyd & Lloyd, 1884).
By this time the U.S., British and many European
pharmacopoeias included H. canadensis as an
official drug. Quantitative methods such as the
Keller-Rusting-Fromme assay were developed,
leading to the commercial availability of
standardized extracts (Motter & Wilbert, 1912).
Given that initially hydrastine was found only in H.
canadensis, it became the key quality marker for
comparing commercial fluid extracts and tinctures,
and in one market survey the majority of products
tested failed to achieve the minimum levels for
hydrastine recommended by the USP (Lancaster &
Davidson, 1927). During the twentieth century
gravimetric, spectrophotometric, chromatographic,
crystallographic and electrophoretic methods were
used to determine the presence of hydrastine and
berberine in H. canadensis (Keenan, 1948; Van
Arkel & Meijst, 1952; Zwaving & de Jong-Havenga,
1972; El-Masry, Korany, & Abou-Donia, 1980).
Using sensitive fluorometric methods developed in
the 1950s, levels of hydrastine could be more
accurately determined (Brochmann-Hanssen &
Evers, 1951).
major alkaloids are shown in Figure 3.
Chemical testing for identity
and purity
Adulteration and substitution of H. canadensis with
Table 2. Potential adulterants of H.
canadensis and their alkaloids.
Species
Berberis vulgaris L.
B. aristata DC.
Mahonia aquifolium
(Pursh). Nutt.
M. nervosa (Pursh).
Nutt.*
Alkaloids
Berberine
Berbamine
Palmatine
Berberine
Oxyacanthine
Palmatine
Coptis chinensis Franch. Berberine
Coptis spp. Salisb.
Epiberberine
Palmatine
Coptisine
Jatorrhizine
Columbamine
Xanthorhiza
Berberine
simplicissma Marshall Jatorrhizine
Rumex crispus L.
None
The advent of liquid chromatography (LC) methods
coupled with a wide array of highly sensitive
spectrometric methods has prompted a new
generation of phytochemists to investigate the
species (Sturm & Stuppner, 1998; Inbaraj,
Kukielczak, Bilski, Sandvik, & Chignell, 2001;
Chelidonium majus L.
Berberine
Chadwick, Wu, & Kinghorn, 2001; Upton, 2001;
Chelidonine
Weber et al., 2001; Li & Fitzloff, 2002; Edwards &
Cheryletherine
Draper, 2003; Brown, Paley, Roman, & Chan, 2008;
Coptisine
Weber, et al., 2003b; Weber & Joseph, 2004; Unger,
Protopine
Laug, & Holzgrabe, 2005; Inbaraj et al., 2006;
Sanguinarine
Gupta, Hubbard, Gurley, & Hendrickson, 2009).
Stypoline
Several new alkaloids have been reported including
berbastine – a second quaternary protoberberine * Mahonia aquifolium and M. nervosa are
however, these are of relatively low concentration.
synonymous with Berberis aquifolium and B.
Gentry et al. (1998) isolated a lactam-containing
nervosa respectively. According to The Plant
alkaloid 8-oxotetrahydrothalifendine. A list of all
List (2010) classification of these species
currently identified alkaloids in H. canadensis is
remains unresolved.
presented in Table 1. Molecular structures of the
6
other berberine-containing species or other
substances of yellow appearance has been going on
since the 1800s (Motter & Wilbert, 1912; Lancaster
& Davidson, 1927; Datta, Bose and Ghosh, 1971;
Govindan & Govindan, 2000). Chemical methods
for validating the identity and purity of H.
canadensis products are still focused on quantifying
berberine and hydrastine levels. The British
Pharmacopoeia set levels for the dried rootstock at
3.0% for berberine and 2.5% for hydrastine (British
Pharmacopoeia Commission, 2006). The USP
further defined goldenseal powdered extracts as
containing not less than 5% hydrastine and 10% total
alkaloids (USP, 2003).
subtracted Raman spectroscopy (SSRS) used for
berberine (Bell et al., 2002). An enzyme-linked
immunosorbent assay (ELISA) using monoclonal
antibodies and linked to a HPLC system has been
successfully used for quantitative analysis of
Tims (2006) has subsequently developed a LC-MS
method that was able to resolve ten analytes
(berbamine, berberine, canadine, chelerythrine,
coptisine, hydrastinine, hydrastine, jatrorrhizine,
palmatine and sanguinarine) from six different plant
species (H. canadensis, Coptis japonica, Berberis
vulgaris, Chelidonium majus, Mahonia aquifolium
and Sanguinaria canadensis), quantitating the
presence of adulterants as low as 5%.
Recently an LC method validated in accordance with
AOAC single-laboratory guidelines for determining
levels of berberine and hydrastine in H. canadensis
has been reported (Brown et al., 2008; Brown &
Roman, 2008). A validated LC/MS method was used
to analyse H. canadensis root from three suppliers
along with the common adulterants (Weber, et al.,
2003a; Weber et al., 2003b). Adopting this HPLC
method, Kamath, Skeels, & Pai, (2009) identified
different alkaloid profiles for H. canadensis and two
berberine-containing Coptis species (see Figure 4).
Other analytical systems have been developed to
detect both major and minor H. canadensis
alkaloids, including capillary electrophoresis-mass
spectrometry used for berberastine, berberine, βhydrastine, canadaline and canadine (Sturm and
Stuppner, 1998); a pH-zone refining counter current
chromatography used for detecting berberine,
canadaline, canadine β-hydrastine and
isocorypalmine (Chadwick et al., 2001); and shifted
Figure 4. HPLC chromatograms of (A) Coptis
trifolia, (B) H. canadensis and (C) C. chinensis.
Coptisine and palmatine are absent from H.
canadensis, while hydrastine is found only in H.
canadensis. Reproduced from Kamath, Skeels, &
Pai (2009). Chinese Medicine – Open Access
Publication, published by BioMed Central
http://www.biomedcentral.com/about/license
7
berberine in H. canadensis and other species (Kim,
Tanaka, & Shoyama, 2003).
Despite the increasing sophistication and versatility
of LC systems, low cost thin layer chromatography
(TLC) methods remain a method of choice within
herb industry quality control laboratories. Datta,
Bose and Ghosh, (1971) described a TLC and ultra
violet (UV) absorption method for authenticating
homeopathic tinctures of H. canadensis. Two spots
present on the chromatograms tested positive for
alkaloids, and the application of chemical reagents
coupled with UV spectrum analysis indicated the
presence of berberine and hydrastine (Datta, Bose, &
Ghosh, 1971). Govindan & Govindan, (2000)
employed TLC to estimate levels of hydrastine and
berberine in ten goldenseal preparations (eight
extracted from authenticated raw samples, two from
commercial capsules containing powder). Results
showed only half of the ten samples tested contained
both alkaloids, in varying quantities and ratios. Four
of the products tested contained only berberine,
while one sample contained neither hydrastine nor
berberine, indicating substitution with less expensive
alternatives. The findings were verified using HPLC
analysis (Govindan & Govindan, 2000).
In their Goldenseal Root Monograph, the American
Herbal Pharmacopoeia (AHP) assert that mere
presence of specific alkaloids is insufficient to rule
out the potential of adulteration with other
berberine-containing plants, but that screening for
alkaloids not found in H. canadensis is equally
important (Upton, 2001). To this end the AHP
adopted a validated high performance TLC
(HPTLC) method that analyses palmatine (found in
common substitutes), along with berberine,
hydrastine and hydrastinine. Hydrastinine is
considered a reliable marker for old or poor quality
H. canadensis, as it is primarily a degradation
product of hydrastine (Upton, 2001). This method
successfully distinguishes H. canadensis from
several potential adulterants (see Table 2).
TLC can also be coupled with mass spectrometry
8
(TLC-MS) for improved analyte detection. The
technology has been used for fast screening of both
high and low molecular weight compounds
including alkaloids (Santos, Haddad, Höehr, Pilli, &
Eberlin, 2004). Van Berkel and co-workers
developed a TLC/desorption electrospray ionization
(DESI)-MS method to analyse a selection of
commercial goldenseal products and standards (Van
Berkel, Tomkins, & Kertesz, 2007). Quantification
of berberine and hydrastine agreed closely with the
values stated on the labels of two products, however
there was marked variation in alkaloid levels of
other products, and the authors concluded that one
product contained alkaloids from an undeclared herb
- thereby implying the presence of an adulterant
(Van Berkel et al., 2007).
Distinguishing H. canadensis
from potential adulterants,
proof of principal
Although HPLC is a useful tool for identifying the
presence of adulterants, HPTLC, a more practical,
affordable and greener technology, is sufficient for
identifying medicinal plants such as H. canadensis
and distinguishing adulterants. Six samples of
ethanolic extracts of H. canadensis radix from
various herb companies were assayed using HPTLC
to distinguish the gldenseal alkaloids (Figure 5). In
this study other berberine containing plant species potential adulterants - were also analyzed for
comparison to H. canadensis (Figure 6).
Figure 5 shows H. canadensis extracts from various
manufacturers. Lanes 1 and 2 illustrate fresh root
extracts, while lanes 4-6 show the dried root
extracts. The reference standards berberine,
palmatine and β-hydrastine can be seen in lane 7. In
spotting the HPTLC plates, the different herb-toextract ratios were accounted for and normalized.
All lanes were loaded with volumes of extract that
accounted for approximately the same mass of plant
material, based on the herb-to-extract ratio of each
extract.
Figures 5A & 5B illustrate the presence of berberine
and hydrastine (although with varying band
densities) in all extract samples analyzed. Figures
5A and 5B also show the absence of palmatine in all
sample lanes. Given the presence of berberine and
hydrastine and the absence of palmatine in these
samples, these data suggest that none of the H.
canadensis extracts studied were adulterated with
other berberine containing plants.
Figure 5B illustrates the HPTLC plate after
derivatization with ninhydrin reagent visualized
under 366 nm UV light. Berberine is seen in all
samples analyzed. Figure 5B also shows a band
above the berberine band (Rf ~0.95) that is of an
unidentified phytochemical. The density of this band
varies significantly from extract to extract, being
absent in the extracts from companies A and F and
having the highest density in the extract from
company B. However, the significance of this
difference in banding densities cannot be interpreted
without identification of the corresponding
phytochemical. Nonetheless, it does illustrate that
there are differences between H. canadensis extracts
produced by various companies.
Figure 6 illustrates the HPTLC analysis of various
medicinal plant species containing berberine:
Mahonia repens (Lindl.) G. Don (lane 1), Berberis
vulgaris (lane 2), Xanthorhiza simplicissima (lane
3), H. canadensis (lane 4) and Coptis chinensis (lane
5). Hydrastine at Rf ~0.3 is clearly present in H.
canadensis (lane 4) but is absent from the other
species.
In addition, palmatine (Rf ~0.4) is present in all
species except H. canadensis. As demonstrated in
these relatively quick and inexpensive assays,
HPTLC can be sufficient and practical for the
identification of H. canadensis and its potential
adulterants. Moreover, a practical approach to the
detection of an adulterant in a H. canadensis lot of
root material would be the detection of palmatine
and hydrastine in the same sample. This would
indicate that the H. canadensis lot has been
adulterated with a berberine containing plant, a
common adulterant strategy.
Materials and methods
Chemicals and Standards: All reagents were
purchased from VWR (Bridgeport, NJ). Standards
were purchased from Chromadex (Santa Anna, CA).
Preparation of material for analysis: Samples of
hydroethanolic extracts of H. canadensis radix were
prepared with a 2:3 (extract:methanol) dilution. To
equalize all extracts to the same concentrations
various root to menstruum ratios were normalized by
adjusting spotting volumes. A confounding issue in
the production of fresh root extracts has been the
substantial water content of fresh plant. Since fresh
root tinctures are generally extracted at 1:2 but this
does not include the water content of the plant
material, we accounted for the additional water from
the root of H. canadensis into our sampling. When
the water content of the root (≈70%) is considered in
these extracts, the actual plant mass (dry equivalent)
to menstruum ratio is 1:9. We therefore utilized a 1:9
ratio to calculate a comparative mass of plant
material for spotting the fresh plant extracts onto the
HPTLC plates.
Chromatographic conditions: HPTLC was
performed on CAMAG (Multenz, Switzerland) precoated silica gel 60 F254 HPTLC plates (10 x 10
cm, 300 µm layer thickness). Samples were applied
to the layers as10 mm-wide bands positioned 10 mm
from the bottom and 8 mm from the side of the plate,
using a CAMAG Linomat 5 automated TLC
applicator with the nitrogen flow providing a
delivery speed of 150 nL/second from the syringe.
After sample application plates were developed in a
CAMAG twin trough glass tank pre-saturated with
the mobile phase EtOAc:MeOH:Formic acid:Water
(50:10:6:3, v/v). The plates were developed to a
distance of 80 mm. The TLC assays were performed
under laboratory conditions of 25 ± 5˚C and 27 %
relative humidity.
9
Detection: After drying (RT, 5 min) the
chromatographic separation was visualized in a
CAMAG UV cabinet (white light, 254 and 366 nm).
A.
1
2
3
4
5
6
7
Post-chromatographic derivatization was performed
with ninhydrin reagent (dried 100 ˚C, 2 min). The
chromatographic conditions had previously been
optimized to achieve the best resolution.
B.
1
2
3
4
5
6
7
Figure 5. HPTLC Plate of Hydrastis canadensis radix ethanol (60%) extracts. From the Herb Pharm
Research and Development Laboratory, Analyst Jean Brown. A) wavelength=254 nm B) Ninhydrin
derivatization, wavelength=366nm.
Lane 1: Company A, fresh root 1:2; Lane 2: Company B, fresh root 1:2; Lane 3: Company C, dry root 1:3;
Lane 4: Company D, dry root 1:4; Lane 5: Company E, dry root 1:5; Lane 6: Company F, dry root 1:5; Lane
7: Reference compounds: berberine (upper), palmitine, hydrastine (lower).
A.
1
2
3
4
5
6
B.
1
2
3
4
5
6
Figure 6. HPTLC Plate of multiple plant species containing berberine. From the Herb Pharm Research and
Development Laboratory, Analyst Jean Brown. A) wavelength=254 nm B) Ninhydrin derivatization,
wavelength=366nm.
Lane 1: Mahonia repens; Lane 2: Berberis vulgaris; Lane 3: Xanthorhiza simplicissima; Lane 4: Hydrastis
canadensis; Lane 5: Coptis chinensis; Lane 6: Reference compounds: berberine (upper), palmitine,
hydrastine (lower).
10
Biosynthetic studies
For many decades scientists have been investigating
possible biosynthetic pathways towards the
formation of isoquinoline alkaloids in plants. Zenk
and co-workers described the biosynthesis of
berberine from the amino acid L-tyrosine via a series
of 13 enzymatic reactions, while (S)-reticuline was
found to be the key branch-point intermediate in the
biosynthesis of individual alkaloids (Zenk, Rueffer,
Amann, Deus-Neumann, & Nagakura, 1985; Rueffer
& Zenk, 1985; Zenk, 1995). This was the first
completely described alkaloid biosynthetic pathway,
with all 13 enzymes identified and named (Otani et
al., 2005). Complementary DNA (cDNA) of several
of the enzymes has been identified and cloned,
opening up prospects for metabolic engineering of
this and other alkaloids (Sato et al., 2001). Berberine
bridge enzyme (BBE) catalyzes the initial step in
converting (S)-reticuline to the quaternary form of
berberine (Bird & Facchini, 2001). Using RNA
interference (RNAi) technology Fujii and coworkers transformed cultured California poppy
(Eschscholzia californica Cham.) cells by downregulating the BBE reaction, which led to
accumulation of the key metabolite reticuline
without influencing cell growth (Fujii, Inui, Iwasa,
Morishige, & Sato, 2006).
Studies on the berberine-containing Coptis japonica
Thunb. Makino reveal the presence of a cDNA
encoding a multidrug-resistance protein (MDR). The
transport protein slows down the influx of berberine
into the cells of rhizomes via an ATP-dependent
process, thereby helping to prevent the accumulation
of toxic levels of the alkaloid (Shitan et al., 2003).
Otani et al. (2005) identified a second transport
mechanism in C. japonica rhizomes, an
H+/antiporter that effluxes berberine rapidly from
the cytosol to the vacuole via a proton gradient.
Since Coptis is also in the Ranunculaceae it is likely
that similar alkaloid transport mechanisms are
present in H. canadensis.
Phenylpropanoids
Chlorogenic acid, neochlorogenic acid – highest in
leaves (McNamara, Perry, Follett, Parmenter, &
Douglas, 2004)
Flavonoids
Using nuclear magnetic resonance (1H- and 13CNMR) spectroscopy, two new C-methyl flavonoids
were identified: 6,8-C-dimethylluteolin 7–methyl
ether, 6-C-methylluteolin 7–methyl ether (Hwang et
al., 2003). In a bioactivity-guided fractionation study
utilizing preparative HPLC, three flavonoids
previously reported in Eucalyptus spp. were isolated:
sideroxylin, 8-desmethyl-sideroxylen, 6-desmethylsideroxylin (Junio et al., 2011).
Organic acids
Quinic acid derivatives, hycandinic acid esters
(Gentry et al., 1998). A newly discovered compound
5-O-(4‘-[β-d-glucopyranosyl]-trans-feruloyl) quinic
acid could serve as a non-alkaloidal marker for H.
canadensis roots (McNamara et al., 2004).
Sterols
β- sitosterol 3-O- β-D-glucoside (Hwang et al.,
2003).
Other constituents
Volatile oil, resin, fatty acids (Upton, 2002).
5. Pharmacology and
Toxicology
Pharmacokinetics
Analysis of human urine following oral
administration indicates the bioavailability of
berberine is quite low, however significant levels of
berberine conjugates have been detected (Yu et al.,
2000; Pan et al., 2002b). A validated LC-MS/MS
method for determining levels of H. canadensis
alkaloids in human serum has been reported.
Berberine was shown to be absorbed more rapidly
11
following oral administration of the H. canadensis
test supplement compared to administration of
isolated berberine (Gupta et. al., 2009).
A Chinese study involving beagle dogs demonstrated
low bioavailability for berberine hydrochloride
following oral administration (Shen, Sun, & Wang,
1993). This finding was subsequently confirmed in
rats, where <5% bioavailability was observed for
berberine (Pan, Wang, Liu, Fawcett, & Xie, 2002).
Berberine and other H. canadensis alkaloids are
substrates for the xenobiotic efflux pump pglycoproteins (P-gp), and observed interactions with
P-gp inhibitors suggests P-gp inhibits net absorption
of berberine in the gut (Pan et al., 2002a). Phase I
metabolites of berberine - known to be biologically
active - have been identified using LC/MS, and
found to remain in the plasma of rats for sustained
periods (Zuo, Nakamura, Akao, & Hattori, 2006). In
vitro microbial transformation of hydrastine using a
fermentation broth model representing human
metabolism has been demonstrated (Herath, Ferreira,
& Khan, 2003).
H. canadensis alkaloids possess
methylenedioxyphenyl (MDP) moieties which
interact with cytochrome P450 (CYP) enzymes to
form complexes (Chatterjee & Franklin, 2003). Both
the root extracts and individual alkaloids inhibited
CYP reactions, particularly the CYP3A4 isoform, in
which hydrastine was more inhibitory than
berberine. CYP inhibitory activity for H. canadensis
extracts was confirmed in humans for both CYP 3A4
and CYP2D6 isoforms, at doses between 2.7g and
3.9g daily taken in three separate doses (Gurley et
al., 2005; Gurley et al., 2008a; Gurley et al., 2008b).
In vitro studies in human liver microsomes indicate
that H. canadensis extract, berberine, hydrastine and
canadine inhibit the CYP2E1 isoform, with
hydrastine the most potent of the alkaloids tested
(Raner et al., 2007). Using an ‘N-in-one cocktail’
method that allows nine major CYP enzymes to be
monitored simultaneously with human liver
microsomes, methanolic extracts of H. canadensis
caused significant inhibition of isoenzymes
12
CYP2B6, CYP2C19, CYP3A4, CYP2D6 and
CYP2E1 (Sevior et al., 2010). The implication of
these collective findings is that ingestion of H.
canadensis could inhibit the metabolism of
pharmaceutical drugs if taken concomitantly
(Chatterjee & Franklin, 2003; Gurley et al., 2005;
Budzinski et al., 2007) (see below). A review of the
pharmacokinetics of H. canadensis alkaloids,
including a proposed metabolic scheme for
berberine, was recently published by the National
Institute for Health (National Toxicology Program,
2010).
Pharmacodynamics
There is a large body of literature concerning
antimicrobial, antiparasitic, antitumor and other
activities associated with berberine (Anonymous,
2000; Mills & Bone, 2000; Grycová et al., 2007).
Quaternary protoberberine alkaloids such as
berberine react with nucleophiles, disrupt cell
membranes, bind to microtubules, act as DNA
intercalators, inhibit DNA synthesis and uncouple
oxidative phosphorylation (Schmeller, Latz-Brüning,
& Wink, 1997; Ball et al., 2006; Grycová et al.,
2007). The ability of berberine to affect so many
molecular targets helps to rationalize the numerous
recorded biological activities for H. canadensis,
however it is evident that other constituents and
mechanisms not associated with berberine are also
involved.
Antimicrobial Activity
In an antimicrobial screening program H. canadensis
extract exhibited significant activity against multiple
drug resistant strains of Mycobacterium tuberculosis,
other Mycobacterium species as well as other human
pathogens. Bioassay-guided fractionation revealed
berberine to be the active constituent, however
berberine alone was less active than the crude extract
(Gentry et al., 1998). Scazzocchio and co-workers
found H. canadensis extracts were active against
both Gram positive and Gram negative strains of
pathogenic microorganisms. In this case canadine’s
activity was similar to berberine, but canadaline
provided the strongest inhibitory activity, especially
of the Gram positive organisms tested –
Staphylococcus aureus (2 strains) and Streptococcus
sanguis - based on killing times and minimal
inhibitory concentrations (MIC values)
(Scazzocchio, Cometa, Tomassini, & Palmery,
2001). Once again the whole-plant extract
demonstrated equivalent or superior antibacterial
activity compared to the individual alkaloids.
Similarly H. canadensis extract inhibited the growth
of resistant strains of Neisseria gonorrhoeae, while
berberine alone was less effective (Cybulska et al.,
2011).
Berberine and two flavonoids from H. canadensis,
but no other constituents, showed inhibitory activity
against oral pathogens Streptococcus mutans and
Fusobacterium nucleatum, the results suggesting one
or more of the flavonoids potentiated the effect of
berberine (Hwang et al., 2003). H. canadensis
extracts also inhibited growth of Helicobacter pylori
in a screening survey of medicinal plants using a
micro-dilution assay (Cwikla et al., 2010).
These studies suggest that while berberine is a key
constituent, on its own it cannot reproduce the
demonstrated antimicrobial properties of H.
canadensis. In fact berberine is regarded as a rather
weak antibacterial at low concentrations (Stermitz,
Lorenz, Tawara, Zenewicz, & Lewis, 2000).
However mechanisms for infection control and relief
of symptoms that do not involve inhibition of
bacterial growth have been established for berberine.
Sack & Froehlich, (1982) demonstrated antisecretory
effects against Vibrio cholerae and E. coli
enterotoxins, providing a rationale for the traditional
use of berberine-containing species in treating
diarrheal disease. Berberine also reduces adherence
of E. coli and Streptococcus pyogenes to epithelial
cells (Sun, Abraham, & Beachey, 1988; Sun,
Courtney, & Beachey, 1988). Reduction of the E.
coli load in the gut by these mechanisms is likely to
also reduce the migration of pathogenic bacteria to
the urinary tract, hence helping prevent urinary tract
infections (Amalaradjou & Ventikanarayanan 2011).
Bacterial resistance
Isoquinoline alkaloid cations are known substrates
for bacterial multi-drug resistant (MDR) efflux
pumps, which in turn effectively pump constituents
such as berberine back out of bacterial cells (Tegos,
Stermitz, Lomovskaya, & Lewis, 2002). However
Stermitz et al. (2000) demonstrated that in the
presence of an MDR inhibiting compound (5’methoxyhydnocarpin {5’-MHC}), found in certain
North American species of Berberis, the
antimicrobial potency of berberine was potentiated
due to increased accumulation in the bacterial cell.
Synergistic combinations of berberine with MDR
inhibitors have been used as models for new
antimicrobial drugs that avoid bacterial resistance
(Ball et al., 2006). With respect to H. canadensis,
extracts of aerial portions (but not the roots) were
shown to contain efflux pump inhibitors, however
this effect was not associated with berberine,
hydrastine or canadine, which are more concentrated
in the roots (Ettefagh, Burns, Junio, Kaatz, & Cech,
2010). Synergism between the berberine and the H.
canadensis aerial parts extract led to enhanced
antibacterial action. Subsequently synergy-directed
fractionation of H. canadensis leaf extracts tested
against Staphylococcus aureus led to the
identification of three flavonoid MDR pump
inhibitors in the leaves (Junio et al., 2011). The
authors conclude that a combination of berberine
containing roots and flavonoid containing leaves
prepared from H. canadensis could produce an
extract with optimal antibacterial activity. Unlike
berberine, canadine does not act as a substrate for
MDR inhibitors (Abidi, Chen, Kraemer, Li, & Liu,
2006).
Fewer investigations have been conducted into
effects on viruses, but in a recent study berberine
inhibited two strains of H1N1 influenza virus in
vitro by several orders of magnitude more than the
anti-influenza A drug amantadine. H. canadensis
extracts were also effective - but only at high
concentrations (Cecil, Davis, Cech, & Laster, 2011).
13
Immunological Activity
Despite the widespread belief that H. canadensis is
an immunostimulant (Chatterjee & Franklin, 2003),
there are few studies to substantiate the claim
(Bergner, 1997). Studies on rats exposed to the
KLH antigen indicate augmented immunoglobulin
(IgM) response for the first two weeks of a six week
treatment period, but failed to improve the IgG
response (Rehman et al., 1999). Berberine has been
shown to inhibit production of TNF-α and the
prostaglandin PGE2 from macrophages induced by
influenza A virus, in an in vitro study (Cecil et al.,
2011).
Anti-cancer effects
Berberine (as berberine sulfate) inhibited the activity
of two tumor promoting compounds in cell cultures
in dose-dependent manner, and produced significant
reduction of the tumor yield and percentage of
tumor-bearing mice (Nishino, Kitagawa, Fujiki, &
Iwashima, 1986). Mechanisms for the purported
anticancer activity of berberine include
complexation with DNA and RNA, inhibition of Nacetyltransferase (NAT) in tumor cells, inhibition of
cycloxygenase II (Cox-2), inhibition of teleromerase
and topoisomerase, antiproliferent effects on tumor
cells, activation of multiple signaling pathways,
antioxidant activity, inhibition of nuclear factor-κB
activation, inhibition of metastases and improvement
of multi-drug resistance (Sun, Xun, Wang, & Chen,
2009).
Little work has been conducted on H. canadensis
itself. In the form of a potentized homeopathic drug
(200C), H. canadensis decreased chemicallyinduced tumors in rats compared to controls, while
decreasing levels of the elevated marker enzymes
ALP, GPT, and GOT (Kumar et al., 2007). In a
separate study the homeopathic ‘mother tincture’
(equivalent to a 1:10 herbal tincture) was highly
cytotoxic in a variety of cancer cell lines, while in
Ehrlich carcinoma cells the 200C potency showed
higher cytotoxicity. The mother tincture and the 30C
potency, but not the 200C potency, induced
14
apoptosis in DLA cells (Sunila, Kuttan, Preethi, &
Kuttan, 2009).
Cardiovascular effects
Investigations involving berberine indicate
numerous beneficial effects on the cardiovascular
system (Lau, Yao, Chen, Ko, & Huang, 2001).
These include positive inotropic, antiarrhythmic,
vasodilatory and antihypertensive effects (Lau et al.,
2001). H. canadensis extract markedly upregulated
low density lipoprotein receptor (LDLR) expression
in HepG2 (hepatic) cells, thereby decreasing
cholesterol and lipid accumulations in plasma and
the liver in vivo (Abidi et al., 2006). In this study the
alkaloid canadine demonstrated greater activity on
LDLR expression than did berberine.
Influence on glucose
metabolism
Berberine has long been investigated for purported
hypoglycemic activity, and two pilot studies in
humans indicate that it may be as effective in Type2 diabetes as the prescribed drug metformin (Yin,
Xing, & Ye, 2008). Various mechanisms of action
have been proposed. Investigations using the Caco-2
cell line indicate berberine decreases glucose
absorption from the intestine by α–glucosidase
inhibition (Pan et al., 2003). Berberine may also
improve insulin action by activating 5' adenosine
monophosphate-activated protein kinase (AMPK) –
a key enzyme in cellular energy homeostasis (Steriti,
2010). When H. canadensis extract was tested on
streptozotocin-induced diabetic mice there was no
influence on blood sugar or insulin levels, however
hyperphagia and polydipsia – symptoms associated
with diabetes – were significantly reduced
(Swanston-Flatt, Day, Bailey, & Flatt, 1989).
Effects on smooth muscle
Many isoquinoline alkaloids have spasmolytic
properties analogous to papaverine. At low
concentrations berberine induced aortic relaxation in
rats by an endothelium dependent mechanism
(Wong, 1998). An H. canadensis alcoholic extract
induced smooth muscle relaxant effects in rat uterus
and guinea-pig trachea. Some but not all of this
activity was attributed to interaction of Hydrastis
alkaloids with β–adrenergic receptors (Cometa,
Abdel-Haq, & Palmery, 1998). Other possible
mechanisms for relaxant effects (on isolated guineapig trachea) include interaction with adenosinic
receptors, K+, or Ca2+ channels, and the releasing of
nitric oxide (Abdel-Haq et al., 2000).
Toxicology
While there is no acute toxicity data available for H.
canadensis, oral LD50s for berberine in mice (329
mg/kg) and berberine sulphate in rats (>1,000
mg/kg) have been published, indicative of high level
of safety when used orally (National Toxicity
Program, 2010). An investigation into acute toxicity
of hydrastine and hydrastinine determined LD50s for
intraperitoneal administration in rats (Poe &
Johnson, 1954). Hydrastine showed ‘a fairly high
toxicity’ but the relevance of these findings to oral
consumption is unclear.
No evidence of fetus malformation or developmental
toxicity was seen in mice or rats in reproductive
toxicity studies conducted by the National Toxicity
Program (NTP) (National Toxicity Program, 2010).
Reproductive screening tests conducted at the
University of Sydney provided contrasting results
between in vivo and in vitro studies (Yao, Ritchie, &
Brown-Woodman, 2005). There was no increase in
pre-or post- implantation losses or any
malformations in female rats given acute doses
equivalent to 65 times the daily human dose of H.
canadensis, however cytotoxic effects were
observed in cultured rat embryos exposed to the
extract. The authors conclude that on the basis of
their in vivo findings, H. canadensis at prescribed
human doses is safe for use during pregnancy, and
the discrepancy with the in vitro findings is most
likely due to poor absorption from the small intestine
(Yao et al., 2005).
In the most recent NTP investigation (TR 562), male
and female rats fed diets containing between 9,000-
50,000 ppm H. canadensis root powder for 2 years
incurred increased incidences of hepatocellular
adenomas or carcinomas. There was also some
evidence of carcinogenic activity in male but not
female mice exposed to the same regime (National
Toxicity Program, 2010). While survival rates in
treated male rats were reduced, treated female rats
had increased survival rates (significantly so at the
9,000 ppm dose p= 0.025) over the 2 years
compared to controls. There was no change in
survival rates for mice. Although the doses that
proved toxic in this study were at the high end of the
equivalent human dose range, clearly long term oral
consumption of the herb should be avoided.
One case study was cited in which an 11-year-old
African American girl with type-1 diabetes who had
taken 1,000-1,500mg goldenseal supplement daily
for at least 2 weeks, presented with diabetic
ketoacidosis (Bhowmick, Hundley, & Rettig, 2007).
No further details of the supplement are given.
Investigations revealed the patient had severe
hypernatremia and hyperosmolality, which the
authors attribute to the herbal supplement. There are
suggestions that H. canadensis acts as an aquaretic,
allowing for excretion of water while sodium is
retained (Chavis, 2003).
Drug interactions
As noted above, in vitro and in vivo studies indicate
H. canadensis inhibits some human cytocrome P450 isoenzymes, potentially influencing the
bioavailability of pharmaceutical drugs taken
concomitantly. One such isoenzyme - CYP3A4 –
metabolizes the anti-HIV drug indinavir. However in
a study of 10 healthy human volunteers H.
canadensis extract had no influence on the
bioavailability of indinavir (Sandhu, Prescilla,
Simonelli, & Edwards, 2003). Despite this finding,
many apparently authoritative sources advise against
the use of H. canadensis while taking indinavir or
other prescription drugs known to be metabolized by
CYP3A4 or CYP2D6 isoenzymes (Cassileth, Yeung
& Gubili, 2010). Unfortunately, at this time many
drug-herb interactions are based on in vitro research
15
7. Modern
Phytotherapy
giving rise to weakly supported theoretical concerns
(Freeman & Spelman, 2008).
Another mechanism by which H. canadensis could
potentially influence drug bioavailability is via
multi-drug resistance (MDR) regulation by berberine
and/or other alkaloids. In one study berberine
induced the MDR transporter Pgp-170 expression in
a range of cell lines, and reduced response of cancer
cells to the cancer drug Paclitaxel (taxol) (Lin, Liu,
Wu, & Chi, 1999).
H. canadensis may also inhibit the actions of
antihypertensive and anticoagulant drugs, and could
potentiate the effects of barbiturates (Cassileth,
Yeung & Gubili, 2010).
6. Clinical studies
Berberine has been subjected to a number of clinical
investigations, however there are few studies with H.
canadensis preparations. There have been several
published studies on the use of berberine for
diarrheal disease coming out of India. These studies
have rather mixed results, and the methodologies of
some have been criticized (Mahady & Chadwick,
2001). In a study of 127 children with moderate to
severe diarrhea, 68.5% responded to berberine
treatment (Chauhan, Jain, & Bhandari, 1970). In a
randomized controlled trial (RCT) of 165 adults with
acute diarrhea linked to E. coli enterotoxins or
cholera assessed by stool volume data, berberine
treated subjects had significantly less diarrhea
compare to controls (Rabbani, Butler, Knight,
Sanyal, & Alam, 1987). In a previous RCT of 185
adults with cholera set in Burma (Khin-Maung-U,
Myo-Khin, Nyunt-Nyunt-Wai, Aye-Kyaw, & Tin-U,
1985) stool volume was also reduced, however
berberine-treated subjects suffered more severe
symptoms compared with those treated with the
antibiotic drug tetracycline.
Modern therapeutic use of goldenseal reflects
pharmacological and clinical research as well as
traditional indications. Naturae Medicina and
Naturopathic Dispensatory (Kuts-Cheraux 1953)
notes many indications due to its healing effect on
Table 3: Modern phytotherapeutic uses of H.
canadensis
Actions
Bitter tonic
Choleretic
Stomachic
Antiparasitic, antiprotozoac
Antimicrobial Antihemorrhagic
Antidiarrheal
Oxytocic
Therapeutic indications
Sinusitis, conjunctivitis, tonsillitis, otitis media,
respiratory infections with catarrhal discharge
Atonic dyspepsia, gastritis, peptic ulcer, anorexia
Diarrhea, colitis, hepatobiliary disorders
Menorrhagia, dysmenorrhea, salpingitis, leucorrhea
Eczema, acne, boils, abscess, pruritis
Protozoal and tapeworm infestations
Varicose veins, phlebitis
Externally for mouth ulcers, wounds and
inflammatory skin disorders, eye baths
In vaginal suppositories for candida infections,
trichomonas and gardnerella
(Bradley, 1992; Mills & Bone, 2000; Skenderi,
2003; Trickey, 2003)
16
damaged mucosa. It is recommended both internally
and externally for ophthalmic disorders such as
conjunctivitis; naso-pharyngeal disorders where the
mucus is thick and membranes swollen;
gynecological conditions such as vaginitis; and
gastrointestinal disorders such as ulcerative colitis.
While acknowledging these uses the British herbalist
Frank Roberts adds that H. canadensis has a unique
action on the venous circulation, namely stimulation
of blood returning to the heart (Roberts, 1978). The
British Herbal Compendium (Bradley, 1992) repeats
traditional indications of “menorrhagia, atonic
dyspepsia and gastritis” but also mentions antimicrobial activity of berberine and other alkaloids.
Other authors and practitioners extrapolate further
from research on the isolated alkaloids and suggest
goldenseal to be of benefit in diarrhea, diabetes,
hypercholesterolemia and infections in general
(Braun & Cohen, 2010). Restoration of mucous
membrane integrity is a key effect for modern
herbalists (Mills & Bone, 2000). While H. candensis
has found popularity amongst the American public
as a cold and flu preventative or treatment, with rare
exceptions (Mitchell, 2003) this use is not supported
by modern herbalists and naturopaths. While Tigler
(2009) does make reference to the colds and flu
indication, she provides in addition this rather more
useful symptom picture: “It is used for atonic
chronic mucosal problems with pale relaxed tissues
as well as sub-acute mucosal mucus membrane
problems with red, engorged tissues” (Tigler, 2009,
p. 97).
Specific indication
Atonic dyspepsia with hepatic symptoms.
Combinations
With Commiphora myrrha (Nees) Engl.and
Echinacea spp. for boils and abscesses
With Achillea millefolium L. and Geranium
maculatum L. for menorrhagia (Trickey, 2003).
With Hamamelis virginiana L. leaf and Euphrasia
spp. as eye lotion (British Herbal Medicine
Association, 1983).
Preparations and dosage
Dried rhizome and root, 0.5-1.2g (Upton, 2001)
Tincture 1:10, 0.3-1mL three times daily (Bradley,
1992)
Tincture 1:5, 8mL daily (American Pharmaceutical
Association, 1946)
Side effects and contraindications
The Botanical Safety Handbook classifies H.
canadensis in Class 2(b): “Not to be used during
pregnancy” and noted the fresh plant may cause
irritation of the mucosa (McGuffin, Hobbs, Upton, &
Goldberg, 1997). According to other sources it should
not be used by neonates due to the potential of
berberine to displace bilirubin from serum protein
(Upton, 2001). Large doses used topically are known
to have caused severe skin irritation or ulceration
(Upton, 2001). The British Herbal Pharmacopoeia
(1983) contraindicated H. canadensis in hypertension,
however that contraindication has been removed in
the more recent British Herbal Compendium (Bradley,
1992).
Regulatory Status
H. canadensis is regulated in the U.S.A. as a Dietary
Supplement.
8. Sustainability and
cultivation
Ecological status
Range: H. canadensis is native to North America, and
can be found growing from Ontario to Arkansas,
across the southeastern U.S. to Georgia, and north to
Quebec. Cultivation projects in Oregon (Oregon's
Wild Harvest, 2012, Baker, 2010), Washington
(Frontier Coop, 2012) and British Columbia (Beyfuss,
2012) have been successful in growing the species
outside of its native range.
17
critically endangered in Tennessee (See Table 4).
For other states within its’ natural range, the lack of
a status listing does not imply that the plant is
abundant. Some states, such as South Carolina,
Louisiana and Florida do not have current estimates
of plant populations (NRCS, n.d.).
It should be noted that the status designations are
continually evaluated and updated.
Harvesting & Collection
regulations
Trade in H. canadensis was well-developed by the
mid-1800s, and until recently most of this trade
was obtained from wild sources. Concern over the
possible unsustainable nature of the industry can be
found as early as the 1800s (Lloyd & Lloyd, 1884).
Figure 7. Hydrastis canadensis - Natural range and
distribution (NRCS, n.d.)
Interest in H. canadensis as a “crop” can be traced
to the early 1900s, as evidenced by a bulletin
produced in 1908 by the USDA (Burkhart, 2006).
Globally H. canadensis is listed as G4: common and
apparently secure (NHPMDNR, 2010), though it may
be rare in parts of its range, especially at the
periphery. According to the Convention on
International Trade in Endangered Species of Wild
Fauna and Flora (CITES, 2012), since 1997 H.
canadensis has been listed in Appendix II which:
Global trade and markets for H. canadensis are
controlled by CITES (US Forest Service, 2010).
At the state level, each state varies in its
governance and control of H. canadensis that is not
intended for export. Not all states estimate plant
populations or control harvesting/trade (see Table
5). Note: state data frequently changes, please
check current state and federal laws before
engaging in any harvesting, selling or buying
activity with H. canadensis.
includes species that are not threatened
presently with extinction but may become so
unless trade is closely controlled. International
trade in Appendix-II species requires a permit
from the exporting country; no import permit is
necessary for these species. Permits are only
granted if the exporting country is satisfied that
certain conditions are met; above all, that trade
will not be detrimental to the species’ survival
in the wild.
H. canadensis is listed as a threatened plant in
Maryland and New York, vulnerable in Pennsylvania,
endangered in Connecticut, Georgia, Massachusetts,
Minnesota, New Jersey, North Carolina, Vermont and
18
Market data: Harvesting impact,
tonnage surveys
Profits from growing H. canadensis vary according
to scale of harvest, yield, source, identity
cleanliness of product and chemical profile
(Burkhart, 2006). Growing H. canadensis under
artificial shade-cloth may result in greater
economic returns due to increases in yield and
efficiency, although quality may also be a factor
(Burkhart, 2006, 2012).
According to Sharp (2003), H. canadensis is one of
the top six selling medicinal plants in the United
States, and has been listed in the official
pharmacopoeias of several European nations,
including France, Germany, Italy and Great Britain.
Every year ten metric tons of H. canadensis roots
are exported to the U. K. alone. In 2006, prices per
pound for dried roots ranged from $35 wild to $50
Table 4. H. canadensis - Status as a rare, threatened endangered plant
Area
Status
Source
Global
G4
Natural Heritage Program Maryland Department of Natural Resources,
(2010)
US-federal
none
US Fish and Wildlife Service, (2012)
Connecticut
endangered
Connecticut DEEP, (2010)
Georgia
endangered
Georgia Department of Natural Resources, (2010); US Fish and Wildlife
Service, (2012)
Maryland
threatened, S2 Maryland Department of Natural Resources, 2010
Massachusetts
endangered
Michigan
threatened, S2 Plants are subject to over-harvesting and habitat destruction. Known in 21
counties, hidden populations are thought to exist in the state (Michigan
Natural Features Inventory, 2004)
Minnesota
endangered
New Jersey
endangered, S1 NJ Division of Parks and Forestry. (2010)
New York
threatened, S2 New York Department of Environmental Conservation. (2012)
North Carolina
Natural Heritage and Endangered Species Program, (2008)
Massachusetts Division of Fisheries and Wildlife, (1994/2010).
At the northern edge of its natural range, H. canadensis is known to exist in
5 Minnesota counties. Wild populations continued to be intensively and
unsustainably harvested by commercial root diggers. Construction and
grazing have also contributed to a loss of local populations and reduced
habitat. (Minnesota Department of Natural Resources, 2012)
As of December 1, 2010, Hydrastis canadensis is no longer listed on the
protected plant list for North Carolina. (North Carolina Department of
Agriculture and Consumer Services, 2010)
Pennsylvania
S4
The Pennsylvania site linked to NatureServe. January, 2012. (Pennsylvania
Natural Heritage Program, nd)
Tennessee
critically
Hydrastis canadensis is listed as one of five plants threatened by
endangered, S3 commercial harvest (Crabtree, 2008)
Vermont
endangered
Note: Vermont offers guidelines for natural plant communities. Vermont
Natural Heritage Information Project (2011)
19
for organic woods-cultivated roots (Burkhart, 2006).
Table 5. Examples of regulation of H. canadensis
by selected states.
From 2006 through 2012 the average price paid for
dried goldenseal root has fluctuated between five and State Regulatory requirements
nine dollars a pound (Burkhart, 2012).
VT Fees are to be charged to a person applying to
Table 6 lists the tonnage for wild and cultivated roots
take a threatened or endangered species as
over a twelve -year period, while average growers
follows: (A) To take for scientific purposes, to
prices over a 42 year period are shown in Figure 8.
enhance the propagation or survival of the
species, or for educational purposes or special
Cultivation:
purposes consistent with the federal Endangered
Species Act, $50.00 (B) To take for a zoological
Soil Requirements
exhibition or to lessen an economic hardship,
H. canadensis thrives best in loamy soil with
$250.00 (State of Vermont, 2010).
abundant organic matter and a pH between 5.5 and
6.5 (Burkhart, 2006; Eldus, 1996; Cech, 2003;
MN Any digging of H. canadensis roots in the wild
Tilford, 1998). Burkart (2006) notes that H.
is illegal in Minnestota without a permit from
canadensis can be found where there is seasonal
the Minnesota Dept of Natural Resources (MN
flooding as well as in seasonally moist upland areas.
DNR, 2011).
Eldus (1996) notes that a 70-80% shaded slope is
preferred. Tilford (1998) suggests a dense hardwood PA Buying, trading, or bartering plants listed as
vulnerable is prohibited within Pennsylvania
canopy on a north-facing hillside. The Natural
without first obtaining a vulnerable plant
Heritage Society of Massachusetts (2010) records
license. This license is granted on an annual
finding H. canadensis in three types of mesic forest:
basis to any interested individual provided
1. an oak/conifer forest dominated by a black birch
he/she complies with record-keeping
canopy, 2. sugar maple forests with Asarum species
requirements. The DCNR oversees this program
and trout lilies and 3. with P. quinquefolium L. under
and uses information. Collection of H.
a sugar maple canopy.
canadensis from state parks in Pennsylvania is
not permitted. Similarly, collecting H.
There are several cultivation methods currently in
canadensis from state game lands in the
use:
Commonwealth is unlawful (Burkhart, 2006).
Forest Farming
H. canadensis collectors and growers in
Woods cultivation has been practiced using raised
Pennsylvania are not required to maintain
beds within a natural or created forest setting. Raised
records of their activities if the product is
beds reduce root competition and result in large wellintended to be sold to a buyer within
formed roots (Burkhart, 2006). This requires more
Pennsylvania or the United States. If the grower
labor and investment to start but may result in
is gathering or producing H. canadensis under
increased yields and better profits. Raised beds may
contract for an exporter, he only needs to
be used to establish plantings for production of seed
maintain records for the benefit of the exporter.
or transplants for shade or wild-simulated
cultivation, however, care must be taken not to erode NC Requires a permit for cultivation. See: Davis &
McCoy (2000)
or otherwise jeopardize the site. To create a 4 foot
raised bed, a grower would need to dig an area 6-8
MI State legislation allows threatened species to be
feet wide using the outer soil to create a mound
taken for propagation, scientific study and
about one foot high. This should be allowed to settle
educational purposes (Michigan State
in order to reduce heaving and plant loss during the
Legislature, 2009).
20
Table 6. Sources of Hydrastis canadensis root and leaf 1999-2010 in pounds (Adapted from AHPA, 2006,
2007, 2012)
* For 1999-2003, for fresh roots, the harvest numbers were extrapolated from tonnage counts in prior years.
For these years, the numbers are rounded, however, the percentage cultivated remains the same.
winter (Burkhart, 2006). Some growers adjust the
pH with limestone (Burkhart, 2006) or potash
(Eldus, 1996). Fertilizers are not recommended.
Some of the disadvantages of this method are a
known increase in the incidence of disease and pests
as well as an investment in materials, time and labor
(Burkhart, 2006).
Wild-simulated
Wild-simulated cultivation involves planting roots,
seeds or transplants in a forest environment with
minimum intervention. This echnique may take from
2-3 years longer to reach harvest and result in a
lower yield when compared to woods cultivation
(Burkhart, 2006). Burkhart maintains that the quality
of the roots is the same between different cultivation
techniques. The most important factors will be
quality of plant material and site selection. A site
may be thinned to create the optimum shade
requirements but should be done without altering the
ecology of the site (Burkhart, 2006). Sites that
contain plants with shallow roots such as
rhododendrons, spruce, hemlock, cedar and other
conifers should be avoided because they may
21
plants.
iv. Companion planting with P.
quinquefolium forming an inner circle and H.
canadensis an outer circle.
Field cultivation
Figure 8. Average price of goldenseal (Hydrastis
canadensis) root and herb (1968–2010) (Burkhart,
2012).
compete for moisture and nutrients, although H.
canadensis has been found growing under conifers
(Eldus, 1996). In late summer or early autumn, the
site is further prepared by brushing the leaf litter
aside, sowing the seeds or planting the transplants
and then replacing the litter (Burkhart, 2006).
Initially the plants/seeds are placed 2-4/square foot
and then thinned, if necessary, to one plant per
square foot. There may be some natural thinning as a
result of animal browsing, insect defoliation or
disease, but H. canadensis is generally not known to
have many pests (Burkhart, 2006; Eldus, 1996). Care
should be taken not to trample the area as soil
compaction may affect the plants. According to
Persons and Davis, (2005), preparing one acre of
land for cultivation requires roughly six hundred
hours of intense labor.
Eldus (1996) describes four methods for wildsimulated planting of H. canadensis. These methods
might also be tried for wood or field plantings.
i. Digging a vertical trench from the top of a
ridge to the bottom of the creek with plants spaced
6-8 inches.
ii. Placing fallen logs about 5-10 feet apart
behind existing trees to create a raised or terraced
area for planting.
iii. Companion planting with Panax
quinquefolium, by alternating with the H. canadensis
22
Field cultivation relies on shade-cloth, lattice or slat
frames to provide the plants with the necessary
amount of shade. Plants grown under 70% shade
showed almost double the overall weight, increased
rootlets and larger leaves compared to plants grown
under full sun (Quigley & Mulhall, 2002). Eldus
(1996) suggests using raised sloped beds to aid
drainage. While shade cultivation may represent an
additional cost, it also allows the grower to monitor
and protect his crop. During the winter plants should
be monitored for exposure from ground heaving
which can expose and kill the roots (Burkhart,
2006).
Plant development
H. canadensis has a series of vegetative stages: the
cotyledon stage (which may last one or more years),
single leaf stage (generally second to third years or
more) during which roots develop and the
reproductive stage (third-fifth year) when flowering
and fruiting occur (Burkhart, 2006). The
reproductive stage is characterized by the
development of an additional 2-3 leaves. Self-fertile
flowers bloom in early spring from March through
May, depending on the location, latitude and soil
condition. Fruits take from 8-12 weeks to ripen
forming a bright red fruit containing one or two
small black seeds (Burkhart, 2006, Cech, 2003;
Eldus, 1996; Blakley & Sturdivant, 1999).
Starting with seed
At least three months of exposure to cold
temperatures (cold stratification) at 35 to 40°F is
required before germination will occur, this cold
treatment also deters fungal disease (Cech, 2002).
Germination may be best when seed and fruit are
planted about 1/2 to 1" deep immediately upon
harvest (Burkhart, 2006). If the seeds are sown in
the fall and mulched, Stoltz (1994) cautions that the
mulch should be removed in spring before the
seedlings emerge.
If the seed is to be collected and cleaned, Burkhart
(2006) suggests that the fruit be soaked in water 2448 hours to soften the fruit and facilitate seed
removal. Eldus (1996) cautions that care must be
taken not to damage the seeds or to allow them to
dry out. Once cleaned, seeds can be stored beneath
3-4 inches of sand or mulch in an outside are where
they are exposed to natural rain (Eldus, 1996) or
refrigerated until planting time. Harvesting and
planting seeds is considered responsible stewardship
as it helps maintain genetic adaption and diversity
(Burkhart, 2006).
Seeds should be planted about four inches apart in
rows about twelve inches apart (Tilford, 1998). At
Tai Sophia Institute, sowing 10 seeds/fruits in
August 2010 resulted in 50% germination by April
2011. All seeds were sown within one foot of the
original plant. Adam (2004) notes that once a field
of H. canadensis is established, the existing stock
should provide seeds and rootlets for new plants.
Planting roots
H. canadensis forms colonies, where many stems
arise from a single interconnected root system
(Burkhart, 2006). A marketable crop may be
produced more quickly (3+ years) from large root
pieces as compared to small root fragments (5+
years). Although small root fragments may
eventually develop into H. canadensis plants, ideally
a grower should divide roots into pieces larger than
1/2 inches with at least one bud. Division is best just
before the plants become dormant during late
summer or early fall. Roots should be planted 2-3
inches deep with the bud oriented upwards. Small
roots may be used to reduce planting costs but these
may remain dormant for a year or more after
planting and will take longer to reach harvest size,
although they may be used to develop plants for
transplant resale (Burkhart, 2006).
Pests and disease
Deer have been known to browse H. canadensis
(Burkhart, 2006; Eldus, 1996). Moles may
sometimes be a problem but can be controlled by
placing wire mesh eight to twelve inches into the
soil (Eldus, 1996).
Under cultivation, several cases of Botrytis (leaf
blight) have been documented (Beyfuss, 2011), as
well as root knot nematodes (Eldus, 1996),
alternaria, rhizoctonia, and fusarium (Davis &
McCoy, 2010). According to Cech (1999), the use
of multi-cropping (P. quinquefolium, Asarum
canadense L.) in cultivated settings will help reduce
the risks of pests and disease. Eldus (1996), Beyfuss
(2011) and Burkhart (2006) noted that diatomaceous
earth may discourage slugs from feeding on H.
canadensis leaves.
Harvesting
When Sanders & McGraw (2005) examined
harvesting practices and subsequent re-growth using
leaf size as an indicator of plant health, they found
that when roots were dug in midsummer before
dormancy, the remaining patches took longer to
recover than when harvesting occurred after the
plants were dormant.
There is some disagreement regarding the potency of
goldenseal roots as the plant ages. According to
Beyfuss (2011) the value of H. canadensis does not
increase with age because as roots become
overcrowded growth slows. For optimum
production, Beyfuss (2011) suggests that plants
should be thinned or spaced after 3-4 years to
prevent overcrowding. Quigley & Mulhall (2002)
suggest that alkaloid concentration may vary
according to the age of the root and/or the speed of
growth, and call for further studies in this area.
During the autumn months alkaloid concentrations
are at their highest, and water content is at its lowest,
suggesting that this is the ideal time to harvest
(Douglas et al, 2010). Harvesting times are
determined by the method of propagation. Plants
23
propagated by division will typically mature in their
third or fourth year, and plants started from seed will
mature in their fifth or sixth (Stoltz, 1994).
Under good growing conditions, one can expect to
harvest between one and two thousand pounds of H.
canadensis per acre (Beyfuss, 2011). Roots should be
thoroughly cleaned, ensuring all dirt, stones and
organic matter are removed and rinsed before drying
(Lockard & Swanson, 2004).
cultivation, and can be quite profitable for growers.
Hence commercial production is possible without
resorting to harvesting of wild populations.
Nevertheless protection and monitoring of wild H.
canadensis sites should remain a high conservation
priority.
Findings from a recent toxicological study by NIH
suggest potential carcinogenicity to rodents when fed
high doses of H. canadensis over a long period (2
years) (National Toxicity Program, 2010). Further
Proper drying from 1-2 weeks will help ensure that
investigations are needed to establish whether these
spoiling, or molding does not occur, and that the roots findings can be replicated in a parallel setting or in
contain the highest concentration of medicinal
alternative models of carcinogenicity.
constituents possible (Cech, 2002; Eldus, 1996).
Roots will lose from 70-75% of their mass during
There is a need for further clinical investigations
drying (Eldus, 1996; Stoltz, 1994). Cech (2002)
(preferably RCTs), and the herbal community is best
suggests drying at 70° F for one day then increasing
served when such studies are designed so to assess
the temperature to 95 ° F, rotating the roots daily until traditional uses. In the case of H. canadensis,
the roots snap. Tobacco barns may be used for bulk
traditional use would emphasize sub-acute infections
drying while for smaller scale production a small shed as referred to in the Modern Phytotherapy section of
or room may be utilized (Davis & McCoy, 2000).
this monograph.
Cech notes that the dried roots are hygroscopic, and
should be protected from moisture and condensation. Coupled with the clinical investigations, research
According to Davis and McCoy (2000), yields from
into the molecular effects of H. canadensis would
shade-grown plants averages around eight hundred
also ideally continue and explore antimicrobial
pounds per acre.
modes of activity, as well as proteomic and genomic
research to further the understanding of this
medicinal plant’s effects on cellular pathways.
7. Summary and moving
forward
In keeping with some other high profile Appalachian
herbs, research into H. canadensis has largely focused
on one or more of its active chemical constituents, in
this case the widely distributed alkaloid berberine, and
to a less extent the alkaloid hydrastine. The common
finding that activities of isolated alkaloids singly or
additively are generally weaker by comparison to H.
canadensis extracts have led some recent investigators
to focus more on potential synergistic activity
between constituents, and results from these studies
are promising (Junio et al., 2011).
Unlike some other ‘at risk’ or endangered
Appalachian herbs H. canadensis is well adapted to
24
Figure 9. Hydrastis canadensis: flower and early
leaves. Photo by Jesse Sommerlatt.
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Conservation Service, US Department of Agriculture. Retrieved from
http://plants.usda.gov/java/profile?symbol=HYDRA2
38
Van Arkel, C. G., & Meijst, M. (1952). Comparative study on determination of hydrastine in Hydrastic
rhizome and liquid Hydrastis extract. Pharmaceutisch Weekblad, 87(49-50), 853-861.
Van Berkel, G.J., Tomkins, B.A., Kertesz, V. (2007) Thin-layer chromatography/desorption electrospray
ionization mass spectrometry: investigation of goldenseal alkaloids. Analytical Chemistry, 79(7),
2778-2789.
Vermont Natural Heritage Information Project (2011) Threatened and endangered plants of Vermont.
Vermont Fish and Wildlife Department. Retrieved from
http://www.vtfishandwildlife.com/wildlife_nongame.cfm
Vermont, State of (2010) Protection of endangered and threatened species. Retrieved from
http://www.anr.state.vt.us/dec/permit_hb/sheet47_4.pdf
Wagner, H., Bladt, S., & Zgainski, E. (1984). Plant drug analysis. New York, NY: Springer-Verlag.
Weber, H. A., & Joseph, M. (2004). Extraction and HPLC analysis of alkaloids in goldenseal. Sheng Wu
Gong Cheng Xue Bao = Chinese Journal of Biotechnology, 20(2), 306-308.
Weber, H. A., Zart, M. K., Ferguson, S. L., Greaves, J. G., Clark, A. P. … Smith, C. (2001). Separation and
quantitation of isoquinoline alkaloids occurring in goldenseal. Journal of liquid chromatography &
related technologies, 24(1), 87-95.
Weber, H. A., Zart, M. K., Hodges, A. E., Molloy, H. M., O’Brien, B. M. … Smith, C.S. (2003). Chemical
comparison of goldenseal (Hydrastis canadensis L.) root powder from three commercial suppliers. J.
Agric. Food Chem., 51(25), 7352-7358. doi:10.1021/jf034339r
Weber, H. A., Zart, M. K., Hodges, A. E., White, K. D., Barnes, S. M. … Harris, R.K. (2003b). Method
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Wong, K.I. (1998). Mechanism of the aortic relaxation induced by low concentrations of berberine. Planta
Medica 64, 756-757.
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Clinical Pharmacology 16, 36-39.
39
Zenk, M. (1995). Chasing the enzymes of alkaloid biosynthesis. Organic reactivity: physical and biological
aspects (pp. 89-109). UK: Newcastle Upon Tyne: Royal Society of Chemistry.
Zenk, M. H., Rueffer, M., Amann, M., Deus-Neumann, B., & Nagakura, N. (1985). Benzylisoquinoline
biosynthesis by cultivated plant cells and isolated enzymes. Journal of Natural Products, 48(5), 725738. doi:10.1021/np50041a003
Zuo, F., Nakamura, N., Akao, T., & Hattori, M. (2006). Pharmacokinetics of berberine and its main
metabolites in conventional and pseudo germ-free rats determined by liquid chromatography/ion trap
mass spectrometry. Drug Metabolism and Disposition, 34(12), 2064 -2072.
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The following WWW sites provide further information related to Hydrastis canadensis.
Pennsylvania Department of Conservation and Natural Resources (DCNR): contains information specific to
goldenseal in Pennsylvania, including publications, educational opportunities, and dealer contact
information. http://www.dcnr.state.pa.us/forestry/wildplant/vulnerable_plants.aspx
Pennsylvania Flora Project (Morris Arboretum,University of Pennsylvania): contains information about the
flora of Pennsylvania, including publications and educational opportunities. http://www.pafl ora.org/
U.S. Fish and Wildlife Service (USFWS): information about CITES and listed species. http://www.cites.org/
U.S. Fish and Wildlife Service (USFWS): contains information about how to obtain a permit to export
goldenseal from North America. http://international.fws.gov/permits/plants.html
40
Appendix 1.
Line drawings of H. canadensis
botanical features. Reproduced
from Bowers, H. in Botanical
Gazette, 1891
41

Goldenseal An Appalachian Plant Monograph Hydrastis canadensis

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